Adult Bone Marrow-Derived Stem Cells for the Lung: Implications for Pediatric Lung Diseases

TIMOTHY VAN HAAFTEN, AND BERNARD THE´ BAUD Department of Pediatrics, Division of Neonatology, Vascular Biology Research Group, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada

ABSTRACT: Bronchopulmonary dysplasia (BPD) and cystic fibro- the “stemness” of the stem cells used are all contentious sis (CF) are two common serious chronic respiratory disorders issues. Furthermore, stem cell research has been markedly without specific treatments affecting children. BPD is characterized slower in lung diseases due to the complexity of the lung. The by an arrest in alveolar growth in premature infants requiring respi- lung possesses numerous anatomical areas (upper airways, ratory support. CF is the most common fatal inherited genetic bronchioles, alveoli and their underlying capillaries) each of disorder characterized by abnormally thick mucus secretions, recur- which contains a unique cellular population with drastically rent infection and ultimately lung destruction. One commonality between these two diseases is the promise of utilizing stem cells different functions. therapeutically. Indeed, the use of exogenous cells to supplement the There are two common, serious pediatric lung diseases natural repair mechanisms or the possibility of genetic manipulation without current treatments that stem cells hold potential prom- in vitro before administration are appealing therapeutic options for ise for: bronchopulmonary dysplasia (BPD) and cystic fibrosis these diseases. Increasing attention has been focused on the use of (CF). BPD is the chronic lung disease of prematurity that adult bone marrow-derived stem cells (BMSC) to regenerate dam- follows ventilator support. BPD is characterized by an arrest aged organs such as the heart, the brain, and the liver. However, due in alveolar and vascular growth (1,2). The long-term conse- to the lung’s complexity as well as the low rate of cellular turnover quences of this halted lung growth are still unknown. There- within the lung, progress has been slower in this area compared with fore, the potential of a stem cell to restore or protect the the skin or liver. Initial work suggests that BMSC can engraft and alveolar epithelium is very attractive as this may reduce the differentiate into a variety of lung cells, but these findings have been morbidity and mortality associated with BPD. CF results from challenged recently. This article critically reviews the current advances on the therapeutic use of stem cells for lung regeneration. mutations in the cystic fibrosis transmembrane conductance (Pediatr Res 59: 94R–99R, 2006) regulator (CFTR) gene and affects 1 in 3200 live births (3). There are five classes of gene defects in CF: 1) defective protein production; 2) aberrant intracellular protein trafficking; he current promise that stem cells hold for tissue regen- 3) defective regulation of chloride transport; 4) reduced chlo- Teration is immense. The belief that once irreversibly ride transport function; 5) reduced protein expression. The damaged tissue can be restored to a normal functional capacity most common cause of CF occurs within class 2 (4). The is providing great optimism for novel treatments in many aberrant intracellular protein trafficking is caused by the de- diseases. However, this research is not without its controver- letion of an amino acid, commonly referred to as ⌬F508 (3,4). sies. There are ethical and moral issues surrounding the use of Pulmonary complications are typically the most serious and stem cells derived from embryos. Recent research is starting to life-threatening manifestations of CF. Major advances in the unravel the potential for adult derived stem cells that was multidisciplinary management of CF have contributed to pro- initially described more than 20 y ago. While the initial results long the life span of affected patients to the mid 30s, but from numerous disease models outside the lung show great quality of life and other CF-related complications justify the potential for a cell replacement therapy, there is heated debate search for a cure. Stem cells have the potential to act as a over numerous concerns within the stem cell field. The mech- vehicle for corrective gene therapy, thereby restoring normal anism of action, the plasticity, the source, the phenotype, and organ function. Ultimately, lung stem cell therapy may also be

Received November 16, 2005; accepted December 14, 2005. Abbreviations: AEC, alveolar epithelial cell; BMSC, bone marrow-derived Correspondence: Bernard The´baud, M.D., Ph.D., Department of Pediatrics, Division stem cell; BPD, bronchopulmonary dysplasia; CF, cystic fibrosis; CFTR, of Neonatology, Vascular Biology Group, HMRC 407, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada, Phone: (780)-492-9479, Fax: (780)-492-3603: cystic fibrosis transmembrane conductance regulator; eGFP, enhanced green [email protected] fluorescent protein; ES cell, embryonic stem cell; GFP, green fluorescent Dr. The´baud is supported by the Canada Foundation for Innovation, the Alberta Heart protein; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; SP and Stroke Foundation (H&SF), the Alberta Hertiage Foundation for Medical Research cell, sidepopulation cell; SP-C, surfactant protein C (AHFMR), the Canadian Institutes for Health Research (CIHR) and the Stollery Chil- dren’s Hospital Foundation. Tim van Haaften is supported by the CIHR-funded Torch (Tomorrow’s Research Cardiovascular Health Professionals) and MFN (Maternal-Fetal-Newborn Health) pro- grams. DOI: 10.1203/01.pdr.0000203550.50258.5a

94R STEM CELLS FOR LUNG DISEASES 95R applicable to other devastating lung disorders such as pulmo- heart, liver, brain, skeletal muscle, and vascular endothelium nary hypoplasia. (15). A true potential for adult derived stem cells lies with the This review will critically analyze the controversial litera- multipotent MSCs and these cells have been extensively used ture investigating the use of exogenously administered stem in lung stem cell research. This potential was first proposed in cells for lung diseases. the 1970s by Friedenstein et al. (16). This initial report showed that plastic adherent cells, previously believed to be DEFINITION OF STEM CELLS committed to a specific tissue type (i.e. bone marrow), had the ability to form cells within the osteogenic, chondrogenic, and Stem cells may be artificially classified into three catego- adipogenic lineages (16). There have been numerous reports ries: embryonic stem (ES) cells, adult bone marrow derived characterizing the phenotype of these MSCs in both humans stem cells (BMSC), and tissue progenitor cells (Table 1). ES (17,18) and mice (16,19). However, it was not until over 20 y cells are pluripotent cells derived from the inner cell mass of after Friedenstein’s first report that the true potential of these blastocyst-stage embryos (5). They are able to form tissue cells were made apparent. In 2001, Krause et al. reported that within each of the germ layers (the endoderm, mesoderm, and a single bone-marrow derived stem cell had the ability to ectoderm) and have thus enormous therapeutic potential to engraft into numerous organs as well as having a “tremendous regenerate any damaged tissue; but ES cells are also mired in differentiative capacity” by adopting the phenotype of epithe- great controversy due to the present need of destroying an lial cells within the liver, lung, GI tract, and skin (20). embryo to harvest pluripotent cells (6). Conversely, progeni- This initial report from Krause has led to extensive study of tor cells are thought to reside within a tissue and are stimu- these cells in the heart (21–24), liver (25–27), brain (28), lated for repair after injury. It is these tissue progenitor cells kidney (29), and endothelium (30). And while these initial that give the liver (the hepatic oval cell) its robust regenerative results show great promise in providing new therapies for ability (7,8). In the lung, type II alveolar epithelial cells (AEC) numerous diseases, these studies have also generated great are the putative distal progenitor cells responsible for repair controversy and highlight the discrepancies that exist within after injury (9,10). Side population (SP) cells, identified by adult stem cell research. These controversies include the their ability to efflux Hoechst dye, and tracheal epithelial basal source and phenotype of the stem cells used, the experimental cells, are putative progenitor cells in the upper airways (11). model of injury, the mechanism of effect, and the methods While the pluripotency of ES and further characterization of used to determine efficacy. While stem cell research in the resident lung stem cells (12,13) hold promise for novel ther- lung has been less prolific than in cardio-vascular or neuro- apeutic approaches for lung diseases, this review will focus on logic diseases, the same controversies apply and will be the adult bone-marrow derived stem cell and their exogenous discussed below. administration in various models of lung diseases. The bone marrow contains several population of primitive cells: 1) Hematopoeitic stem cells (HSCs); 2) mesenchymal STEM CELL THERAPIES TO PREVENT stem cells (MSCs); 3) endothelial stem/progenitor cells LUNG INJURY (EPC); 4) SP cells; and 5) multipotent adult progenitor cells The 2001 report from Krause et al. opened the door to the (MAPC). Among these, the HSCs are the best characterized of potential that there may be an ethically appealing option in the adult stem cells (14). HSCs are multipotent and have the using stem cells for tissue replacement. Based on their previ- ability to maintain or restore the mature circulating blood cells ous work showing the potential of CD34ϩ linϪ for regener- (erythrocytes and leukocytes). Recent evidence suggest that ating hepatocytes (27), Krause et al. extended their observa- bone-marrow derived cells have the ability to cross lineage tion to the lung. Myelo-ablation was induced in female mice barriers and generate differentiated tissue beyond their own by total body irradiation of 1050–1100 cGy (an otherwise tissue boundaries to form functional components of other lethal dose if the bone marrow is not reconstituted; in most tissues, expressing tissue-specific proteins in organs such as cases of irradiation the lungs are also injured). CD34ϩ linϪ cells from male donor mice were administered intravenous Table 1. Terminology (IV) (20). Fluorescent in situ hybridization (FISH) to track the Y-chromosome showed 20% of AEC being derived from a Term Definition Example donor animal. Donor derived BMSC persisted up to 11 mo Totipotent Able to form any cell type, Fertilized oocyte, zygote post-transplant (20). includes the embryo and the trophoblast of the Consistent with this first observation, Kotton et al. reported 6 placenta that plastic adherent MSCs (1–2 ϫ 10 injected IV) from Pluripotent Able to form cells within Embryonic stem cells transgenic mice overexpressing lacZ, allowing tracking of the any of the germ layers: cells via X-gal staining, were able to engraft and form type I the endoderm, ectoderm, AEC in experimental lung fibrosis induced by intratracheal and mesoderm Multipotent Able to differentiate into Mesenchymal stem cells, (IT) bleomycin (31). Bleomycin is one of the most extensively a select range of cell hematopoietic stem cells, studied and reproducible experimental models for lung fibro- types, heavily influenced certain tissue-specific stem sis. When bleomycin is given into the airway, it produces lung by the microenvironment cells, side-population epithelial injury, followed by an inflammatory response over cells several days, followed by lung fibrosis that eventually resolves 96R VAN HAAFTEN AND THE´ BAUD (32). Engrafted cells had morphologic features of type I AEC from the bleomycin-induced inflammation, as assessed by (i.e. flattened with ovoid nuclei bulging into alveolar lumen collagen deposition, and matrix metalloproteinase (MMP) and adjacent to type II AEC) and expressed type I AEC activation (40) as compared with animals that received MSCs specific markers such as Lycopersicon esculentum lectin and one week following bleomycin, indicating the importance of T1␣ (31). The proportion of donor derived MSCs contributing timing of stem cell therapy, as observed in myocardial infarct to the type I AEC population was not assessed. Interestingly (41). The low numbers of donor-derived cells engrafting the however, all bleomycin injured-mice (4/4) showed engraft- lung did not appear sufficient to account for the therapeutic ment when compared with PBS injected controls (2/9), sug- response, suggesting that donor stem cells may have other gesting that prior injury amplifies stem cell engraftment, as local effects. has been observed in other tissues (7). Furthermore, there Rojas et al. used a model in which before bleomycin were no donor-derived type II AECs, even in lungs harvested injury, the animals also underwent myelosuppression via a 1 and 2.5 d postinjection. In vitro, 10% of plastic adherent single administration of busulfan (42). Myelosuppression in- donor bone marrow cells cultured for one week expressed T1␣ creased the susceptibility to bleomycin injury, suggesting that and aquaporin 5, used as markers for type I AEC, (but are not an intact bone marrow serves to limit the extent of lung injury. solely confined to these cells) (33–35), but not surfactant After this suppression of the bone marrow, plastic adherent protein C (SP-C), a type II AEC specific marker (36) suggest- and immuno-depleted MSCs were given IV MSCs adminis- ing that cultured bone marrow cells can serve as type I AEC tration improved survival when compared with untreated an- precursors (31). imals. Furthermore, within the MSC administered group, These two reports form the prototypes for most of the there were a “substantial number” of donor cells within the experiments on BMSC in the lung that have followed. In lungs 2 wk following bleomycin insult with characteristics 2002, Theise et al. reported that donor-markers for either of AEC I and II, fibroblasts, and endothelial cells. In the whole bone marrow or CD34ϩ linϪ cells were detected in bleomycinϩbusulfan group, 29% of cells were derived from 14% of type II AEC after 1200 cGy total body irradiation (37). donor MSCs. Interestingly, circulating levels of granulocyte- Engraftment was still present 6 months later (for total bone macrophage colony-stimulating factor (GM-CSF) and granu- marrow injection) and 8 mo later for CD34ϩ linϪ cells, locyte colony stimulating factor (G-CSF) (factors known to detectable by FISH. In another study, administration of stimulate stem cell mobilization from the bone marrow), were MAPC to nonobese diabetic/severe combined immunodefi- higher in bleomycin-injured animals receiving MSC. This ciency (NOD/SCID) mice following mild irradiation of 250 suggests that, besides lung engraftment, humoral factors may cGy accounted for 4% of the alveolar epithelium assessed by also contribute to stem cell-induced tissue protection. This is ␤-galactosidase immunofluorescence (38). In another elegant consistent with the greater protection conferred by stem cell study, Abe et al. generated parabiotic mice by surgically transplant in bone marrow sufficient (no busulfan) animals. joining green fluorescent protein (GFP) transgenic mice and Preliminary data in oxygen-induced experimental BPD in wild-type littermates (39). These mice develop a common newborn rats also suggest a protective role of MSC. Rats circulation (approximately 50% green cells in blood) by 2 wk exposed to 95% oxygen from birth to 14 d postnatal during the after surgery. The wild-type mouse was either uninjured or alveolar period of lung development have an irreversible irradiated or received IT elastase (a well accepted emphysema arrest in lung development (43). In this preliminary study, model) or the combination of radiation with IT elastase injec- MSC were given IT, a situation that is clinically relevant since tion. Radiation or the combination of radiation with elastase premature infants at risk are routinely given surfactant via this significantly increased the proportion of bright green cells in route and which has been used successfully with other exper- the lungs of wild-type mice. These cells resembled morpho- imental therapies (44). CFSE (a green fluorescent marker)- logically, interstitial monocytes/macrophages, subepithelial fi- labeled MSC given at 4 d postnatal in hyperoxic-exposed broblast-like interstitial cells, and type I AEC. Approximately animals prevented stunted alveolar growth, engrafted and 5 to 20% of lung fibroblasts primary cultured from injured expressed the type II AEC specific marker SP-C (unpublished wild-type mice expressed GFP. data). Ortiz et al. used plastic adherent and purified, magnetic These studies demonstrate the potential of BMSC to home bead immunodepleted MSCs from male bleomycin-resistant to the injured lung, engraft, and adopt the phenotype of one or BALB/c mice expressing CD34, CD45, and CD11b in bleo- many of the lung cells. These reports also illustrate the mycin (4 U/kg)-induced pulmonary fibrosis in female bleo- numerous methods that exist in culturing and selecting BMSC mycin-sensitive C57BL/6 recipients (40). The purified MSCs (Table 2). The differences in experimental design and the (5 ϫ 105) were injected into the jugular vein. Lung engraft- numerous techniques to determine treatment efficacy (engraftment quantified by real-time PCR showed that male DNA ment, differentiation, improvement in lung histology) further accounted for 2.21 ϫ 10Ϫ5% of the total lung DNA in render the interpretation of these results difficult. control-treated mice but prior injury with bleomycin increased Furthermore, there are numerous studies that do not support engraftment by 23-fold. FISH revealed that engrafted male the plasticity of adult stem cells in different organs. Wagers et cells were localized to areas of bleomycin-induced injury and al. attempted to determine the fate of “prospectively isolated, exhibited an epithelium-like morphology, suggesting that stem long-term reconstituting HSCs” by using either a chimeric cells homed specifically to sites of injury. Furthermore, im- animal, produced via bone marrow ablation and reconstitution mediate administration of MSCs was able to protect the lung with GFP HSCs, or using a parabiotic model, joining the STEM CELLS FOR LUNG DISEASES 97R

Table 2. Characteristics of adult bone marrow-derived cells used in lung stem cell biology Cell type Phenotype Hematopoietic Stem Cells CD34ϩ/Ϫ, Stem cell antigen (SCA)-1ϩ, Thy1ϩ, c-kitϩ Lineage (lin)Ϫ—this is the absence of numerous markers for mature lymphocytes (CD45, CD3, CD4, CD8), myeloid (CD11b/Mac-1), and erythroid (TER-119) cells Mesenchymal Stem Cells no unique phenotype adherence to tissue culture plastic main isolation aid CD34ϩ linϪ MSCs (20) Plastic adherent MSCs (31) CD34ϩ linϪ MSCs (37) multi-potent adult progenitor cell (MAPC) (38) Side Population Cells (Hoechst dye efﬂux) (39) MSCs immunodepleted for CD34, CD45, and CD11b (40) MSCs immunodepleted for CD34, CD45, and CD11b (42) Total Bone Marrow, MSCs not isolated (46) Total Bone Marrow, and HSC side population cells (Hoechst dye efﬂux) (47)

circulation of a transgenic GFP mouse with the circulation of This result led to retrospectively analyze the lungs from mice a wild type mouse (45). While the BM ablated group showed receiving actin-eGFP bone marrow transplantation using de- robust reconstitution of circulating HSCs, and the parabiotic convolution microscopy. Deconvolution microscopy was able animals had robust hematopoietic chimerism, there was little to generate a three dimensional image of the lung allowing to evidence for stem cell engraftment and transdifferentiation. determine that the co-expression of SP-C and eGFP was a Only one cell of 13.2 ϫ 106 cells examined within the brain, false positive: the pro-SP-C signal resided just outside the and seven cells of 4.7 ϫ 105 cells examined within the liver eGFPϩ cells, with a difference between the two signals less co-expressed the donor marker GFP as well as a tissue specific than 300 nm, and this phenomenon was not apparent until the marker (albumin for hepatocytes) in the BM ablation group three dimensional image was viewed from multiple angles (45). There was no evidence for engraftment and transdiffer- (46). entiation in the lung. Moreover, the parabiotic model had no Kotton et al. also used SP-C-eGFP mice, but investigated indication of HSC engraftment and transdifferentation in any both unfractionated BM as well as BM-derived SP cells (47). of the tissues analyzed (45). SP cells were purified from the femurs, tibias, and iliac crests In addition, two recent reports have challenged the engraft- and identified using a model of Hoechst dye efflux. These cells ment potential of adult-derived stem cells in the lung. These are enriched for HSC activity. Adult mice underwent two studies take advantage of transgenic mouse that express a myeloablation via either a single dose of 11 or 12 Gy radiation marker protein ubiquitously or under the control of the type II or two doses of 7 Gy (total of 14 Gy). The bone marrow was AEC specific promoter SP-C (46,47). Chang et al. trans- then reconstituted with either a whole bone marrow transplant planted bone marrow (2.0 ϫ 107 cells) from transgenic mice or transplantation of the highly enriched HSCs SP cells expressing the LacZ or eGFP gene ubiquitously (under the from SP-C-eGFP donor mice. Three months post-transplanta- control of the actin promoter), or under the control of the tion, with robust blood reconstitution, the mice were chal- human SP-C promoter into irradiated, neonatal mice. The lenged with a bleomycin injury (0.05 units) and engraftment lungs of recipients transplanted with bone marrow from trans- was assessed 1 mo post-transplant. Using three antibody genic mice that ubiquitously express eGFP or LacZ showed independent assays, fluorescent activated cell sorting cells whose morphology and location were compatible with (FACS), fluorescent microscopy, and real time PCR, the type II AEC (46). These green cells also co-localized with authors showed no evidence of donor cells becoming type II pro-SP-C. These initial results indicated that the eGFP BMSC AECs (47). were contributing to the alveolar epithelium. Flow cytometric In humans, BMSC repopulation also seems to contribute analysis of SP-C immunostained cells with eGFP indicated minimally to the type II AECs after cross-gender lung trans- that eGFP-fluorescent cells accounted for 50–55% of all lung plantation (49). Sequential immunohistochemistry and FISH cells, as well as 70% of type II AECs 2 wk after bone marrow performed on lung biopsy specimens from male recipients of transplantation (46). One month post-transplantation, eGFPϩ transplanted lungs from female donors showed Y-chromo- cells only comprised 2-25% of total lung cells with 0% to 8% some–containing type II AEC in 9 of 25 biopsy specimens of type II AECs being donor-derived. However, upon analysis from five of seven gender mismatched male lung transplant with cyto-centrifugation, the phenotype of the eGFPϩ SP-Cϩ recipients, that accounted for 0% to 0.553% of type II AEC cells displayed a small, round cell type with a scant cytoplasm (49). The number of type II AEC of male karyotype showed which is not congruent with the normal type II AEC pheno- a statistically significant relationship to the cumulative num- type of larger, cuboidal cells with an abundant cytoplasm (48). ber of episodes of acute cellular rejection. This study also Moreover, mice that received SP-C-eGFP bone marrow trans- suggests that BMSC contribute minimally to the type II AEC plant showed no eGFP expression, indicating that the trans- proliferation that is often present in these patients as a sequela planted cells are not adopting the type II AEC phenotype (46). to alveolar injury (49). 98R VAN HAAFTEN AND THE´ BAUD STEM CELL THERAPIES TO REPLACE DEFICIENT numbers of colony-forming units (CFUs) compared with con- GENES IN THE LUNG trol MSCs. ⌬F508 corrected MSCs showed improved chloride efflux in vitro. Furthermore, after co-culture with AECs, the The use of stem cells for CF is still at the conceptual stage, corrected ⌬F508 MSCs were able to secrete a significantly therefore there are currently very few reports on the use of larger amount of chloride when compared with uncorrected adult derived stem cells for CF. However, the idea that adult MSCs from ⌬F508 CF patients. Corrected ⌬F508 MSCs derived stem cells could be used as a vehicle for gene therapy responded to IBMX and forskolin (adenylate cyclase activa- is very appealing. Firstly, this method of administration would tors) to a larger degree than the uncorrected ⌬F508 MSCs give the physician a controlled method of administering the (52). While this in vitro proof of concept study is very corrected gene, circumventing some of the problems that have exciting, definitive answers cannot be concluded until this is been seen using viral mediated gene therapy (50). Secondly, a tested in vivo in an animal model of CF. stem cell could potentially provide a synergistic mechanism. There is one report to date attempting to use adult BMSC Not only would the stem cell deliver the corrected CFTR gene for CF in vivo. In this report, Loi et al. used both plastic to the lung of a CF patient, but these cells could also help adherent MSCs and total bone marrow harvested from trans- replenish destroyed tissue. genic mice over-expressing GFP (53). The authors subse- Grove et al. first showed the potential for a marrow derived quently ablated the bone marrow of CFTR knockout mice cell as a vehicle for gene delivery to the pulmonary epithelium using 800 rads total body irradiation and injected either the (51). MSCs were expanded in vitro after harvest from the bone MSCs (106 cells/mouse) or total bone marrow (20 ϫ 106 marrow by being grown in a defined basal bone marrow media cells/mouse) via the tail vein. They also used an intraperito- supplemented with human IL-6 (IL-6), murine IL-3, murine neal dose of naphthalene (275 mg/kg) either three days before stem cell factor, and polybrene. These cells were then trans- MSC administration or one month after bone marrow recon- duced with a retroviral vector (MND-eGFP-SN) that has been stitution with total bone marrow cells to induce lung injury optimized for long-term expression of eGFP in other cell lines. and stimulate migration of the transplanted bone marrow cells Following transduction, the eGFP marrow stem cells were to the lung. Stromal marrow cells were able to engraft into the then injected via the tail vein into mice that had received 600 alveolar epithelium and to confer CFTR mRNA to the knock- cGY of radiation for 2 consecutive days. This resulted in 1% out mice, but the engraftment occurred at such a low rate to 7% of AEC derived from donor eGFP stem cells (51). (0.025% chimeric airway epithelial cells, of which 0.01% More recently, the possibility of transforming in vitro expressed CFTR protein) that it is unlikely that this method MSCs from CF patients with a viral transcript to correct the would correct the trans-epithelial chloride deficit. Naphtha- defective CFTR gene was reported (52). MSCs were first lene-induced airway remodeling did not significantly increase obtained from healthy volunteers as well as CF volunteers the number of chimeric airway epithelial cells expressing with the ⌬F508 homozygous mutation. MSCs expressing CFTR. GFP, co-cultured with human airway epithelium, adopted a These two reports highlight the potential for BM-derived cuboidal or columnar shape similar to epithelial cells, as well stem cell based gene therapy in CF and the need for new as the expression of cytokeratin-18 (CK-18) a marker of strategies for enhancing airway epithelial engraftment to exert epithelial cells. When directly mixed with primary human a beneficial effect. AECs, control MSC expressed the tight junction protein oc- ϩ cludin. Moreover, FACS was used to isolate the GFP cells CONCLUSION from the co-culture system. These cells, when co-cultured with normal human AEC or with AEC from ⌬F508 homozy- The use of stem cells in the lung is at the crossroads of gous patients, expressed wild type CFTR, which were either understanding. Over the last decade, numerous studies have not detected in MSCs before or that were not co-cultured. demonstrated the ability of adult BM-derived cells to differ- These experiments strongly suggest that MSCs, when exposed entiate into non-hematopoietic lineages, including neural, to AECs, are able to adopt the phenotype of AECs and show skeletal muscle, hepatic, cardiac and vascular lineages and to the importance of the microenvironment in inducing tissue- repair these tissues. With regard to lung repair, the literature is specific transdifferentiation. While these results are very sparser. Initial promising reports showing adult BM-derived promising and lend credence to the ability of MSC transdif- cell homing, engraftment, and lung phenotype adoption are ferentiation into AEC, the true potential for MSC in CF are as now challenged. This highlights the need for more reliable vehicles for gene delivery. MSCs from 3 ⌬F508 homozygous techniques and assays to assess adult BM-derived cells lung patients were transduced with a viral vector for expression of engraftment and transdifferentiation. Other challenges before CFTR (vesicular stomatitis virus glycoprotein pseudotyped moving to the clinical application with this promising thera- Moloney murine leukemia virus-CFTR-neo). In this construct, peutic strategy include: improved characterization of putative the human CFTR gene was linked by internal ribosome entry BM-derived stem cells, determining mechanisms of homing, site to the neo gene to allow for selection of the corrected engraftment and transdifferentiation, as well as the alternate MSCs based on G418-resistance (a sulphate antibiotic) and mechanisms of action beyond pure tissue replacement, rigor- drug-resistant stem cell expansion. Insertion of the gene had ous assessment of therapeutic success combining lung histol- no effect on the multitpotency (based on adipogenic, chondro- ogy and function, and lastly and more importantly determining genic, and osteogenic cell differentiation), doubling times, and the long term safety of these MSCs. The potential for terato- STEM CELLS FOR LUNG DISEASES 99R mas and other cancers is too high a risk without certain 28. 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